This study was approved by the Institutional Animal Care and Use Committee of Shanghai University of Traditional Chinese Medicine.
Neonatal cardiac myocyte culture and treatment protocol
Cardiac myocytes were obtained from the ventricles of 1-day-old Wistar rats and prepared using a modified method as described previously [8]. The cardiac myocytes were plated in DMEM/F-12 (1:1) with 10% horse serum and 5% fetal calf serum. After 48 hours, the cardiac myocytes were incubated in serum-free DMEM/F12 supplemented with 1 ug/mL insulin, 5 μg/mL transferrin and 0.1 mmol/L BrdU. The cardiac myocytes were then treated with norepinephrine (NE, 1 μmol/L) for 72 hours in the presence or absence of carvedilol (CAR, as a positive control, 1 μmol/L) or STA (10 μmol/L), whereas the controls were cultured in medium without treatment.
Chemicals and reagents
Chemically pure STA was obtained from the chemistry laboratory at the Institute of Chinese Materia Medica, Shanghai University of Traditional Chinese Medicine. It was identified on the basis of chemical and spectroscopic evidence using the general formula C7H13NO2. The structure of STA is illustrated in Figure 1[9]. Carvedilol was purchased from the Boehringer Mannheim Company. All chemicals were of analytical grade or the best grade commercially available.
Cell surface area analysis
The cardiac myocytes were washed in Ca2+- and Mg2+-free PBS and fixed in 3.7% paraformaldehyde-PBS (Fisher, Fairlawn, NJ, USA) for 10 minutes at room temperature, then washed in 0.1% Triton X-100 for 5 min. The cells were double-labeled with DAPI (for DNA nuclear labeling) and phalloidin (for F-actin labeling) conjugated with Alexa Fluor 488 (Invitrogen, Carlsbad, CA, USA). Fluorescently labeled cells were then viewed using a fluorescent microscope (Axiovert 40 CFL, Carl Zeiss, Germany). The cells within each field were outlined, and the two-dimensional area was obtained using I-solution software (IMT i-Solution Inc., Daejeon, Korea). The final mean surface area of each group was determined using 150 cells from 3 independent experiments.
Total protein/DNA content ratio analysis
The cells were collected using trypsin/EDTA, and then split into two equal aliquots for protein and DNA measurements. One aliquot was resuspended in protein lysis buffer (50 mmol/L Tris–HCl, pH 7.4, 2 mmol/L EDTA, 1% Nonidet P-40, 100 mmol/L NaCl, 100 mmol/L sodium orthovanadate, and 0.1 mmol/L PMSF with Complete protease cocktail (Roche, Meylan, France)). The protein concentration was determined using a BCA protein assay (Pierce, Rockford, IL, USA). The other aliquot was resuspended in TEN (10 mmol/L Tris–HCl, pH 7.4, 1 mmol/L EDTA pH 8.0, and 150 mmol/L NaCl). The DNA concentration was determined using Hoechst 33258 (Sigma-Aldrich, St. Louis, MO, USA) with calf thymus DNA as a standard (Sigma-Aldrich, St. Louis, MO, USA), as previously described [10]. The DNA was analyzed at 365 nm excitation and 460 nm emission using a fluorescence spectrometer (F-4500, Hitachi, Tokyo, Japan). For each well, the result of protein determination was divided by the DNA measurement to provide a protein/DNA ratio.
Measurement of fetal gene expression
Cells grown on 30 mm Petri dishes were harvested by scraping. The total RNA was isolated using Trizol (Invitrogen, Carlsbad, CA, USA), according to the manufacturer's instructions, and the concentration was determined using a BioPhotometer (Eppendorf, Hamburg, Germany). Complementary DNA was synthesized from 2.5 μg of total RNA using a PrimeScript RT reagent Kit (TaKaRa, Dalian, China). Real time PCR was performed in triplicate and detected by SYBR Premix Ex Taq™ Kit (TaKaRa, Dalian, China) using the LightCycler2.0 System (Roche, Basel, Switzerland). The relative mRNA expression levels were normalized to 18 s ribosomal RNA. The primers were designed by the web based program, Oligo Perfect™ Designer (Invitrogen). The primers used were as follows: 18 s rRNA forward, 5'-ACGGACCAGAGCGAAAGCAT-3'; 18 s rRNA reverse, 5'-TGTCAATCCT-GTCCGTGTCC-3'; α-MHC forward, 5'-GACACCAGCGCCCACCTG-3'; α-MHC reverse, 5'-ATAGCAACAGCGAGGCTCTTTCTG-3'; β-MHC forward, 5'-GGAGCTCACCTACCAGACAGA-3'; and β-MHC reverse, 5'-CTCAGGGCTTC-ACAGGCATCC-3'.
Ca2+-Transient analysis
Free cytosolic calcium was measured in neonatal rat cardiomyocytes as previously described [11]. Cardiomyocytes were plated onto glass coverslips and cultured for 72 hours. Myocytes were loaded with fura-2/AM (Molecular Probes, Eugen, OR, USA) by incubating the coverslips for 30 minutes in 2 mL of Tyrode’s solution containing (mmol/L) NaCl 137, KCl 5, glucose 15, MgSO4 1.3, NaH2PO4 1.2, HEPES 20, and CaCl2 1, as well as fura-2/AM (3 mmol/L) and D-Pluronic (Molecular Probes, Eugen, OR, USA) (3 mL of 25% [wt/wt] in dimethyl sulfoxide). Myocytes were then rinsed with Tyrode’s solution and maintained for 30 minutes at room temperature to allow for deesterification of the dye. Coverslips were transferred to a temperature-regulated chamber (RC-21BRFS, Warner, Hamden, CT, USA) mounted on a Carl Zeiss Axio Observer. A1 microscope, Lambda DG-4 monochromator and filter sets(Sutter, Novato, CA, USA), and cells were perfused with prewarmed modified Tyrode’s solution. Cells were paced by electrical field stimulation (SIU-102, Warner, Hamden, CT, USA) at 1 Hz, 18 V, 4-millisecond pulse duration using platinum electrodes. Fluorescence of intracellular fura-2 was determined by alternatively illuminating cells with 340- and 380-nm light and measuring emission at 520 nm using an EMCCD (iXon Ultra 897, Andor, Belfast, BT, UK). The sampling rate for collection of ratio values was 100 Hz. Images were recorded and analyzed using the MetaFlour software (Molecular Devices, Sunnyvale, CA, USA). As previously reported, improper calibration of fura-2 is difficult to exclude because of compartmentalization in loaded cells and differences in spectral properties between cells and buffer solutions [12]. So the fura-2 fluorescence ratio was used as an indicator of free cellular calcium. Diastolic fluorescence ratio, peak systolic fluorescence ratio, and transient decay (τ, ms) were used for analysis of transient amplitude.
Measurement of SR Ca2+-ATPase activity
The SR membranes from cardiac myocytes were prepared using a modified procedure, as previously described [13]. The cardiac myocytes were homogenized in a buffer containing 300 mmol/L sucrose, 1 mmol/L PMSF, and 20 mmol/L PIPES. The homogenates were centrifuged at 500 g for 20 min and at 25000 g for 60 min to pellet the SR membranes. The SR Ca2+-ATPase activity was measured using a modified method, as previously described [14]. Homogenate protein was added to the solution containing 200 mmol/L KCl, 20 mmol/L HEPES, 10 mmol/L MgCl2, 3 mmol/L PEP, 0.6 mmol/L NADH, 1 mmol/L EGTA, 7.5 U/ml PK, and 5 U/ml LDH (pH 7.0), 2 μM of the Ca2+ ionophore A-23187 (Sigma-Aldrich, St. Louis, MO, USA). NADH was measured using a photometer (DU-640, Beckman Instruments, Inc., Fullerton, CA, USA) with a wavelength of 340 nm at 37°C, and the reaction was started with 1 mmol/L Na2ATP. Absorbance changes were monitored for 3 min. The Ca2+ ionophore was added to prevent intraluminal Ca2+ accumulation in SR, which is inhibitory to Ca2+-ATPase activity [15]. All experiments were performed in triplicate. SERCA2 activity was calculated in nanomoles of ATP per milligram of protein per minute from the change of absorption at 340 nm divided by the extinction coefficient of NADH, in milligrams of protein per minute. Basal activity was measured in Ca2+-free buffer in the presence of EGTA (4 mmol/L), followed by increasing Ca2+ concentrations.
Western blotting
The cells were rinsed with cold PBS and then scraped in ice-cold lysis buffer with protease and phosphatase Inhibitor Cocktail (Roche, Meylan, France). The protein concentration was assessed using a BCA Protein assay (Pierce, Rockford, IL, USA). Then, an equal amount of protein was separated using a 4-20% SDS-PAGE and transferred to PVDF membranes (Hybond, Amersham, Arlington Heights, IL, USA). The blots were incubated with the appropriate antibodies, including anti-SERCA2a, anti–phospho-PLN (serine-16, threonine-17) and anti-PLN (Santa Cruz Blotechnology, CA, USA). The signals were detected using Super Signal Immobilon Western Chemiluminescent HRP Substrate (Millipore, France) and quantified by densitometry using the Image analysis system (Bio-Tanon, Shanghai, China). The data were normalized using β-actin to ensure equal loading and were expressed as a ratio of the experimental group to the control group.
cAMP assay
The concentration of cellular cAMP was carried out using the cAMP-Glo assay kit (Promega, Madison, WI, USA). Briefly, cells were cultured in poly-D-lysine-coated, white, clear-bottom 96-well plates (Greiner, Stonehouse, UK) in a density of 104 cells/well. Cells were lysed to release cAMP, and then the cAMP Detection Solution, which contains protein kinase A (PKA) and kinase substrate (Leu-Arg-Arg-Ala-Ser-Leu-Gly), is added. The Kinase-Glo Reagent is next added to terminate the PKA reaction and detect the remaining ATP via a luciferase reaction. Plates are read using a microplate-reading luminometer (Synergy 2, BioTek, Winooski, VT, US). Relative luminescence output is inversely proportional to cAMP levels.
PKA assay
Protein kinase A (PKA) activity was performed using the ProFluor PKA assay kit (Promega, Madison, WI, USA). Briefly, cell lysates were added to the reaction buffer containing a bisamide rhodamine 110 peptide substrate and were incubated for 30 min at 25°C. The reaction was stopped by adding the termination buffer, which contained a protease that removes amino acids specifically from the non-phosphorylated substrate and results in the production of highly fluorescent rhodamine 110. Thus, the fluorescence intensity is inversely correlated with kinase activity. PKA activity was determined from the fluorescence intensity of the nonphosphorylated substrate using a Synergy 2 microplate-reader (BioTek, Winooski, VT, US).
Statistics
Data are presented as the mean ± standard error of mean (SEM). The intergroup variation between various groups was measured by one-way analysis of variance (ANOVA) with Bonferroni’s post hoc test. Results were considered statistically significant when P values blow 0.05. Data were analyzed using Statistical Package for Social Science (SPSS, Chicago, IL, USA) software version 14.0 for Microsoft Windows.